Phosphoinositides, such as PI(4,5)P 2 (PIP2), regulate various aspects of actin dynamics and vesicle trafficking. However, the precise contributions of PIP2 to vesicle trafficking, its localization, and regulation by synthesis is not well-understood. The synapse offers a good model in which to explore these questions. Therefore, the goal of this project is to explore the presynaptic roles of the PIP2 in synaptic vesicle trafficking and actin dynamics. To do so, I will examine the consequences of altering their availability in living synapses. PIP2 availability will be altered by injecting into lamprey giant reticulospinal synapses either specific, PIP2 binding protein modules to decrease endogenous PIP2 or by injecting reagents that perturb PIP2-metabolizing enzymes. A combination of fluorescence imaging, electrophysiology, and electron microscopy will be used to examine the consequences of these reagents on actin dynamics and synaptic vesicle trafficking in vivo. This approach will provide the first comprehensive analysis of PIP2 functions at the synapse. The De Camilli lab has already identified and characterized enzymes that metabolize PIP2 at the synapse. Genetic and acute impairment of one of these enzymes, the PIP2-phosphatase synaptojanin, alters actin cytoskeleton and disrupts synaptic vesicle recycling in synapses, suggesting an important role for PIP2 in these processes. Altered phosphoinositide metabolism has been implicated in the pathogenesis of Alzheimer's disease, as well as a variety of human disorders of hypertension, glucose metabolism, and lipid metabolism. Thus, my studies may have wide implications for human health.